As the levels of halogenated organics (alleged carcinogens) found in municipal water supplies increase, the risk to public health also increases;therefore, the need to develop ways to degrade these materials is of utmost importance. Bacterial multicomponent monooxygenases (BMMs), enzymes typically consisting of a hydroxylase, a reductase and a regulatory protein, have recently been studied as bioremediation agents. Understanding aspects of BMM catalysis is thus essential for the development of enzymes for the degradation of pollutants. Although methane monooxygenase has now been studied for several decades, the mechanism of proton and electron delivery to the active sites of BMMs is still somewhat of a mystery. With the help of the recent crystal structure of phenol hydroxylase (PHH) and its regulatory protein (PHM) we have proposed several mutations to address these issues. When bound to PHH the regulatory protein, and potentially the reductase, covers an extensive hydrogen-bonding network (conserved in BMMs) that starts at the surface of PHH and extends into the active site. Mutation of residues involved in the hydrogen-bonding network will enable us to determine the role of this network in proton transfer, electron transfer, and structural integrity of the PHH active site. We will monitor the effects of the mutations on the catalytic activity and stability of PH. Alkene monooxygenase (AMO) from R. Rhodochrous B-276, a BMM capable of epoxidation of alkenes, will be cloned into an E. coli vector developed for the expression of multisubunit enzymes. This will facilitate the first structural studies on AMO. Typically the BMM hydroxylases have an a2[unreadable]2?2 heterodimeric composition, but AMO only has an a and [unreadable] subunit. Because there is so much variability in the location and fold of the ?-subunits in BMMs, and AMO performs reactions with high stereo- and regioselectivity in comparison to its BMM counterparts, we believe that the ?-subunit may be playing an important role. PUBLIC HEALTH RELEVANCE Because bacterial monooxygenases are capable of the degradation of a wide range of wastewater pollutants, the potential to engineer these enzymes to be better suited for this role is of great interest. Understanding the mechanisms of O2-activation and substrate specificity in these enzymes from this work will both advance fundamental knowledge and further the development of these enzymes as biocatalysts for wastewater treatment purposes.